The completion of Human Genome Project in 2003 (International Human Genome Sequencing Consortium (2004) “Finishing the euchromatic sequence of the human genome,” Nature 431: 931-945) signaled the beginning of a new era of biomedical research and clinical practice in which biological processes could be studied in unprecedented detail. The current goals of genome research include determining the hereditary factors in disease, developing new methods to detect disease and guide therapy (van de Vijver et al. (2002) “A gene-expression signature as a predictor of survival in breast cancer,” New England Journal of Medicine 347: 1999-2009), and improving the understanding of individuals' metabolisms to accelerate drug discovery. In order to pursue these goals, it will be useful for scientists and clinicians to compare the genetic heterogeneity of countless individuals' genomes. However, sequencing a single human genome can prohibitively expensive and time-consuming. The routine sequencing of individuals' genomes will become a possibility only with the availability of faster and cheaper sequencing technologies.
Sequencing approaches that substantially improve throughput at a reduced cost over classical sequencing methods have been developed. For example, zero-mode waveguides (ZMWs) are powerful new sequencing tools that facilitate detection of labeled single nucleotides into single nucleic acids (in real time) as the nucleic acids are copied by a polymerase (Levene et al. (2003) “Zero Mode Waveguides for Single Molecule Analysis at High Concentrations,” Science 299: 682-686). Efficient DNA synthesis occurs only at substrate concentrations much higher than the pico- or nanomolar regime typically required by other single molecule sequencing technologies. ZMWs overcome this limitation by confining reaction volumes to the zeptoliter range, thereby enabling an inversely proportional increase in the concentrations of DNA sequencing reagents.
Current methods for preparing nucleic acid templates are not optimal for use in high throughput DNA sequencing systems. Conventional cloning and cell culture methods are time consuming and expensive. Lengthy nucleic acid purification protocols currently in use do not reliably produce nucleic acid samples that are sufficiently free of sequencing reaction inhibitors such as salt, carbohydrate and/or protein. Furthermore, these problems are magnified when such conventional techniques are scaled to the quantities that would be useful for high throughput sequencing technologies. Consequently, there is an increasing demand for efficient, low-cost methods for the preparation of high-quality nucleic acid templates. The present invention provides methods and compositions that would be useful for supplying high throughput DNA sequencing systems with such templates.